The fusion of the sperm cell with the oocyte cell generates a new individual containing half of their genetic material from the maternal parent and half from the paternal parent. Mitochondrial genomes, however, exhibit strict maternal inheritance even though both cells contain mitochondria upon fusion. Autophagic destruction of sperm mitochondria after fertilization may facilitate this strict maternal inheritance of mitochondria. When assisted reproductive technologies (ART) are used, the normal process of cell fusion is often bypassed. Especially in the case of intracytoplasmic sperm injection (ICSI), the issue has been raised of whether the sperm organelles are processed in the same manner as they are in conventional fertilization. Since ICSI is now used in the majority of IVF procedures in this country, more research is needed to gain a better understanding of the basic mechanisms that govern the processing of sperm material after fertilization. The nematode, C. elegans, provides an excellent model system for studies on fertilization. Prior studies in C. elegans have provided important insight into the process of fertilization. For example, a GFP:ubiquitin fusion protein localizes to sperm organelles within minutes after sperm-oocyte fusion. This ubiquitination is followed by localization of the autophagy marker, LC3, leading to the lysosomal degradation of mitochondria. These studies revealed that another sperm organelle, the MO, is also destroyed after fertilization using this process. Specific Aim #1 of thi proposal will use a genomic RNAi screen in C. elegans to identify genes required for ubiquitination of sperm organelles. Genes that show loss of GFP:Ub localization upon knockdown will be candidates for components of the pathway leading to ubiquitination sperm organelles. In Specific Aim #2, genes that are identified in the screen as required for ubiquitination will then be used to determine if ubiquitination is required to prevent inheritance f paternal mitochondrial genomes. A PCR test will be used to determine if loss of ubiquitin localization to sperm organelles results in the retention of paternal mitochondria. If such a scenario turns out to be true, we will use these conditions to test the potential negative effects f paternal mitochondrial inheritance. Specific Aim #3 will investigate the role of the proteasome in the inheritance of sperm organelles. Sperm contain high concentrations of proteasomes, but very little ubiquitin. Proteasome inhibition in sperm will be followed by assessment of organelle inheritance after fertilization. This research will advance the understanding of how sperm organelles are processed in the zygote.